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Horizontal Tabs
PDB ID |
Structure Details |
Novel structure of hFBG-C |
|
Group deposition of 11 fragment bound hFBG-C structures |
Protein Expression and Purification
Construct Details
hFBG-C FL (TNCA-c025):
Ile1974—Ala2201 + N-terminal, TEV-cleavable hexahistidine Twin-strep-tag®
hFBG-C ΔCTL (TNCA-c042):
Pro1979—Gly2196 + N-terminal, TEV-cleavable hexahistidine tag
hFBG-C FL Bio (TNCA-c049):
Ile1974—Ala2201 + N-terminal, TEV-cleavable hexahistidine tag + C-terminal AviTagTM
hFBG-C ΔCTL Bio (TNCA-c050):
Pro1979—Gly2196 + N-terminal, TEV-cleavable hexahistidine tag + C-terminal AviTagTM)
Accession: P24821 Isoform 1 (NM_002160.3) + Natural Variant E2008Q.
Expression Cell Line: BL21(DE3) Tuner + CyDisCo pMJS226
Vectors Used: pNIC-NHStIIT, pNIC28-Bsa4, or pNIC-Bio3
Sequence Details
Bold = tag sequence
Bold + Italics = removed by TEV protease
Underlined = hFBG-C
>TNCA-c025 (hFBG-C FL)
MHHHHHHSSGASWSHPQFEKGGGSGGGSGGSAWSHPQFEKGSGVDLGTENLYFQSMIGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRA
>TNCA-c042 (hFBG-C ΔCTL)
MHHHHHHSSGVDLGTENLYFQSMPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEG
>TNCA-c049 (hFBG-C FL Bio)
MHHHHHHSSGVDLGTENLYFQSMIGLLYPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGRRKRASSKGGYGLNDIFEAQKIEWHE
>TNCA-c050 (hFBG-C ΔCTL Bio)
MHHHHHHSSGVDLGTENLYFQSMPFPKDCSQAMLNGDTTSGLYTIYLNGDKAQALEVFCDMTSDGGGWIVFLRRKNGRENFYQNWKAYAAGFGDRREEFWLGLDNLNKITAQGQYELRVDLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYSGTAGDSMAYHNGRSFSTFDKDTDSAITNCALSYKGAFWYRNCHRVNLMGRYGDNNHSQGVNWFHWKGHEHSIQFAEMKLRPSNFRNLEGSSKGGYGLNDIFEAQKIEWHE
Protein Expression and Purification Method
hFBG-C constructs were transformed by heat shock into chemically competent BL21(DE3) Tuner E. coli cells, carrying the ChlorR CyDisCo helper plasmid pMJS226 which was a gift from Prof. Lloyd Ruddock and allows the soluble over-expression of disulphide-containing proteins in the cytoplasm.14 Overnight cultures were inoculated 1:100 into “TB-PLUS” media (1X Terrific Broth + 15 mL/L glycerol + 0.01% Antifoam + 1 mM MgSO4 + 10 mM (NH4)2SO4 + 0.5% (w/v) glucose + 1X E. coli trace metals) and grown at 30°C with 250 RPM shaking to OD = 2.5. Expression was induced with 0.5 mM IPTG and allowed to proceed overnight at 18°C. For biotinylated constructs (TNCA-c049 and -c050), 1 g/L of D-biotin (Fluorochem) was added at time of induction. Cells were harvested by centrifugation, lysed by sonication in Lysis Buffer 1 or Lysis Buffer 2 (biotinylated constructs), and clarified by centrifugation (> 15,000 x g, 60 min). For the first affinity step, either TALON® SuperflowTM (TNCA-c042, -c049, -c050) or StrepTactinXTTM (TNCA-c025) resin was used essentially according to the manufacturer’s recommendations, and hFBG-C was eluted with Elution Buffer 1 (TALON®) or Elution Buffer 2 (StrepTactinXTTM). N-terminal tags were removed via TEV cleavage (either 4 hours at room temperature or overnight at 4°C) and the cleavage efficiency (100%), biotinylation efficiency (>99%), and intact mass were confirmed by LC-MS according to standard methods.18 Finally, the protein was polished on a Superdex 75 16/60 column into Gel Filtration Buffer, and the monodisperse, monomeric peak was concentrated to up to 30 mg/mL before flash freezing in LN2 and storage at -80 °C.
- Lysis Buffer 1: 50 mM HEPES pH = 7.5, 300 mM NaCl, 5% glycerol, 1X benzonase, 1X protease inhibitors
- Lysis Buffer 2: 50 mM HEPES pH = 7.5, 300 mM NaCl, 5% glycerol, 1X benzonase, 1X protease inhibitors, 4 mM D-biotin
- Elution Buffer 1: 50 mM HEPES pH = 7.5, 300 mM NaCl, 5% glycerol, 150 mM imidazole
- Elution Buffer 2: 50 mM HEPES pH = 7.5, 300 mM NaCl, 5% glycerol, 50 mM D-biotin
- Gel Filtration Buffer: 20 mM HEPES pH = 7.5, 250 mM NaCl, 2% glycerol
Disulphide Bond Validation by Intact Mass Spectrometry
To verify the presence of two disulphide bonds in purified hFBG-C, three samples were prepared for intact mass analysis. The first “as purified” Sample A was prepared and analysed as above. The second “+ iodoacetamide” Sample 2 was prepared by denaturing 5 µg of protein for 1 hour in 50 µL of Denaturing Buffer (8M Urea in 50 mM Ammonium Bicarbonate pH = 8.0), followed by the addition of 25 mM Iodoacetamide and a second 1 hour incubation prior to intact mass analysis. The third “+TCEP iodoacetamide” Sample 3 was prepared as for sample 2, except 10 mM TCEP was added to the Denaturing Buffer to reduce disulphide bonds prior to alkylation with iodoacetamide. Each alkylation event corresponds to a mass shift of 57-58 Da, representing the capping of a single Cys residue. In protein with 2 properly formed disulphide bonds, an approximately 230 Da mass shift occurs in Sample 3 while no mass shift occurs in Sample 2 (all relative to as purified sample 1). hFBG-C preps were only used if the expected two disulphide bonds were observed.
Differential Scanning Fluorimetry for Calcium Binding
hFBG-C is known to bind Ca2+ at a mapped “DXD” cation binding loop (D2128—D2130). To validate this activity, we implemented a simple DSF assay essentially as described previously.19 An 11-point serial dilution of CaCl2 was prepared in water at 10X the desired final concentration. In a 96-well v-bottom white qPCR plate, 10 µL of 2X HEPES-buffer saline (HBS) was mixed with 4 µL of water, 2 µL of the 10X CaCl2 dilution series, and 2 µL of protein at a stock concentration of 3 mg/mL (final concentration 0.3 mg/mL). Samples were incubated for 15 min at room temperature before the addition of 2 µL of 10X SYPROTM Orange and a second 10 min incubation in the dark. Melt curves were collected on an MX3005p RT-PCR Machine (Stratagene) from 25°C to 95°C with a ramp of 1°C/min. Melt temperatures were calculated by nonlinear fitting to the ideal monophasic ProteoPlex model,20 and Kd’s were extracted by plotting Tm shifts (relative to protein in the absence of Ca2+) against log[Ca2+] and fitting to a one-site saturation binding model in GraphPad Prism.
Crystallization and Fragment Screening
Throughout, sitting-drop vapour diffusion experiments were performed in 96-well, three-sub-well SWISSCI plates. The structure of hFBG-C (PDB: 6QNV) was solved using a crystal which appeared after 28 days at 20°C with hFBG-C ΔCTL at 17 mg/mL mixed with Morpheus Condition C09 (0.09M NPS + 0.1M Buffer System 3 pH = 8.5 + 50% Precipitant Mix 1).21 Crystals were flash frozen in LN2 and data was collected at Diamond Light Source and automatically indexed, integrated, and scaled using the Xia2 pipeline.22 The structure was phased by molecular replacement using Phaser (search model PDB: 1FIC),23 followed by manual re-building in Coot and refinement in phenix.refine.24,25
For fragment screening, hFBG-C ΔCTL was concentrated to 30 mg/mL and mixed 100 nL : 100nL in all sub-wells of a SWISSCI plate using the custom fine screen “JAC-MorpheusC09-z001”, which samples the chemical space around Morpheus C09 by varying the pH of Buffer System 3 from 7.0-9.0 and the concentration of Precipitant Mix 1 from 44% to 56%. After 14 days, > 150 crystals per plate were obtained and used to complete a full fragment screen of the DSI-Poised 1 Fragment Library (Enamine) at the XChem Facility, Diamond Light Source Beamline i04-1 according to standard methodologies (https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening.html). Followed by automatic data collection, data were imported to XChemExplorer,26 phased by molecular replacement using Dimple,27 and putative fragment binding events were identified by PanDDA.28 Structures were manually triaged and 11 conclusively bound co-crystal structures were refined with REFMAC before deposition.29
Assays
Previously published, detailed step-by-step methods for the NF-κB reporter assay in THP1 BlueTM monocytes and the ELISA in primary human macrophages were followed as recommended.16,17 Anti-TLR4 polyclonal antibody and isotype control were obtained from InvivoGen (PAb-hTLR4; https://www.invivogen.com/pab-htlr4). The final concentration of LPS in the cellular assays was strictly regulated to be < 10 pg/mL to avoid LPS-induced pro-inflammatory signalling. For hFBG-C FL expressed as a soluble protein as reported above, the final purified protein was concentrated to 30 mg/mL and LPS was removed with Thermo Scientific High Capacity Endotoxin Removal Resin according to the manufacturer’s recommendations. Alternatively, endotoxin-free hFBG-C can be prepared by intentional formation of inclusion bodies via expression in standard BL21(DE3) E. coli cell lines, followed by purification, endotoxin removal, and refolding according to previously reported methodologies.8 While we routinely obtain endotoxin-free hFBG-C using our method for use at 2 µM or below, care must be taken to ensure that the requisite LPS threshold is strictly maintained for the specific application and final assay concentration.
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